INTENTIONAL PUBLIC DISCLOSURE
The information contained within this page is intentionally disclosed in lieu of an attempt at reserving intellectual property. All information provided below is free and open source to anyone with the properly regulated facilities who wishes to use it to fight Coronavirus (COVID-19).
Note: This is a page about developing a cheap diagnostic test and does not contain medical advice. Please visit the WHO website for medical advice on COVID-19. If you are experiencing serious COVID-19 symptoms, please call your local emergency number (e.g. 000, 911, etc.)
BioFoundry’s mission has always been to empower humanity through the free access of knowledge and the open-source production of essential medicines. The emergence of COVID-19 has created chaos in an already strained medical systems and exposed the limitations in global diagnostics throughput.
While countries scramble to upscale their medical testing capabilities, this demand is quickly pushing the price for tests beyond many nations’ purchasing power. It may become necessary to develop reliable-alternative COVID-19 diagnostic tests that can provide proper testing to the developing world. Until a functional vaccine or cure is found, testing will be our most powerful tool for containing the spread of the disease. If governments continue to focus on testing within their national borders, we will fail to address the development of hotspots overseas and face an even longer chill in international trade and travel.
Reliable self-diagnostic test is yet to make its way into the hands of patients – forcing everyone to rely on an overburdened medical system with limited thermocyclers for diagnosis. In areas without access to quantitative thermocyclers, let alone the niche enzymes necessary to run a test in these devices, this challenge becomes insurmountable.
BioFoundry proposes the creation of cheap test kits for novel Coronavirus which can be used in place of RTqPCR or MRI scans. This would necessitate a top-bottom supply chain of the necessary enzymes and, ideally, would by thermocycler free, or at least remove the quantitative requirement.
How do we currently diagnose COVID-19?
The trick to detecting viruses is to amplify viral RNA by first converting it into DNA using an enzyme named “Reverse Transcriptase”. The DNA strands produced by this enzyme are known as complimentary DNA (cDNA). Generally, this is done using a quantitative thermocycler, which amplifies the DNA through heating and cooling while simultaneously observing the reaction to see at which stage exponential amplification occurs.
China amazed the world with its portable MRI scanners and 20 minute diagnostic test that searched for specific structures within the lungs, but it’s far too late to start manufacturing enough of these to service the world’s population in time. They will likely also remain beyond the capacity of many governments to purchase en masse. They’re an excellent solution, but difficult to implement NOW.
How will the Open Source diagnostic kit work?
The open-source (OS) community proposes ditching the expensive capital equipment in favour of a reaction that requires only a set of biological and chemical reagents in a single tube that gives a reliable diagnosis. We are currently sourcing the reagents to replicate the experiments being shared within the community. BioFoundry proposes setting up the facilities to synthesise the required biological reagents at scale, while collaborating with our sister company Argent Scientific to source the required chemicals. We would then focus on producing cheap test kits to fill the diagnostic gap in Australia, before moving our focus to Africa and South-East Asia to help support their containment efforts as well.
Loop-mediated isothermal amplification (LAMP) is the primary methodology being proposed to solve this challenge. This link is just one of several publications already in this space, further boosted by a veritable flood of blog posts by contributing scientists. As we build out this resource, we will try to include as many references as we can to the work of these brilliant people.
We ask that you please don’t attempt to recreate these experiments unless your lab is fully authorised by the government. If you have permission from your relevant local authorities and are working on a LAMP diagnostic kit, you can add your data to our public google drive folder.
Step 1: Recreate the Diagnostic Test using a commercial LAMP kit and a positive control and the primers described in OS papers.
This is the stage we are currently at. We have reviewed the existing publications and have decided on a couple of interesting primer sets. To begin testing whether this will be an effective diagnosis, we have decided to use an NEB LAMP kit. This will not be a scalable method, but will help minimise the risk of variables while we test the protocol.
The major challenge we will face during Step 4 will be in scaling our own enzyme production to meet the standard of this kit. For now, we only need to discover quickly whether this protocol will work and be reliable as a diagnostic.
Alongside the kit we will also need primers specific to COVID-19. These are short fragments of DNA that are used in conjunction with the kit to produce DNA strands (amplicons) of a known size. The sequences of these primers and their amplicons are described below. But first, lets have a look at the sequence of the Coronavirus and where the amplicons will be located within the overall genome of COVID-19. All sequences are 5′ to 3′ and the images are courtesy of SnapGene’s software, a long term supporter of our lab.
Note: The sequences shown are for the cDNA produced by the Reverse Transcription Polymerase. The original sequences inside the virus will be single-stranded RNA and all T’s will be substituted with U’s.
Loop F CTGCACTTACACCGCAA
Loop B GTAGCTGGTTTTGCTAAATTCC
619 bp Amplicon: CTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTGTTAAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACCG
ORF1a Amplicon: CCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGTGGTGAGAC
GeneN Amplicon: ATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCA
Step 2: Confirm kit works with positive control. Attempt to source enzymes independent of commercial kit or synthesise in house. Move onto self experimentation.
Positive control is also “on the way” – I don’t want to overpromise when I’m not sure supply chains can deliver. We will see if it arrives in a timely fashion. We’ll post summaries of the labwork here once it starts (ETA: 25th)
Step 3: Seek out a collaboration with the government and a pathology clinic to test our diagnostic kit on a larger spread of the population.
If you know someone, please let them know about us!
We’re already in discussion with several groups at different levels of the Australian government.
Step 4: Scale production of diagnostic kits through the biosynthesis of requisite enzymes. Meet Australia’s diagnostic gap.
* section under construction*
This has long been a plan of Biofoundry, to synthesise these critical molecules with a cheap and open source platform. This would finally allow the decentralisation of the organic molecules industry that is 30 years overdue and the major reason for the testing shortage. In the meantime, we hope to ensure Australia has enough tests to be constantly monitoring the population for infected individuals.
|Reagent in NEB Kit||Is it OS? OS Alternatives?||Efficient & scalable in-house production methods;||External Suppliers|
|Isothermal Amplification Buffer:||1X Buffer Components|
20 mM Tris-HCl – Yes
10 mM (NH4)2SO4 – Yes
50 mM KCl – Yes
2 mM MgSO4 -Yes
0.1% Tween® 20 –
aka. polyoxyethylene sorbitan monolaurate
pH 8.8 @ 25°C
|Best to source chemicals or precursors from the chemical industry.||Our sister company Argent Scientific regularly orders all of these reagents from China. If an alternative supply chain is necessary, Australia has some domestic capacity for the production of these chemicals;|
Tris-HCl – mix from Tris base (expensive) and Hydrochloric Acid (much cheaper)
MgSO4 – cheap and easy to order
Polyoxyethylene Sorbitan Monolaurate – harder to manufacture and order
|Bst2.0 Warmstart Polymerase||No, Several alternatives, but might be able to sort out agreement with NEB to use their product.||Efficient and scalable by anyone with a bioreactor. Breweries can be used in a pinch to improve national capacity. Plenty of alternative open source polymerases that can be used in place of Bst2.0.|
Polymerases are normally an easy purchase from suppliers, but are likely to be in short supply during the coming months.
Potentially partner with UNSW or CSIRO to rapidly upscale production. Need a plasmid first.
|WarmStart RTx Reverse Transcription Polymerase||No, Several alternatives, but might be able to sort out agreement with NEB to use their product.||Efficient and scalable by anyone with a bioreactor. Breweries can be used in a pinch to improve national capacity. There are some open source alternatives for open source RT polymerases, but less so than with regular polymerases.||RT-Polymerases are normally an easy purchase from suppliers, but are likely to be in short supply during the coming months. |
Potentially partner with UNSW or CSIRO to rapidly upscale production. Need a plasmid first.
This is just a basic summary of how a group could obtain the independent
Isothermal Amplification Buffer
Bst2.0 Warmstart Polymerase
Warmstart RTx Reverse Transcription Polymerase
Additional Materials and Equipment Required for Scaled Test Kit Production
Lb media, antibiotic, primers, positive control, pure water, sterile throat/nose swabs, heat block, bioreactors
|Reagent/Equipment||Cost/X||Regular Method to Produce/Obtain||Alternative Method to Produce/Obtain|
|RT Pol Plasmid|
|Sterile Nose/Throat Swabs|
Our sister lab in Melbourne also has ambitious plans in this space. Head over their website to get in contact with Andrew if you’re interested in learning more.
Step 5: Limit testing and ongoing monitoring of kit effectiveness
This kit is useless if it gives false positives or false negatives. This will likely only be able to be determined through wide screen testing alongside a trusted diagnostic test like RTqPCR.
Step 6: Attempt to address diagnostic gap in hard-hit regions with low purchasing power. Attempt to subsidise expenses through purchasing-power based discretionary pricing.
If a vaccine is not developed and implemented in a miraculous period of time, containment of the disease overseas is likely to become of peak national security concern. As such we hope to leverage the Australian Government’s interest in containing the disease to help sell our diagnostic kits at an affordable price overseas. We are happy to consult with investors and the public sector about the ideal business model to effectively distribute this diagnostic test. While all our research will be OS, we must ensure we create an effective business model to achieve efficient distribution. What matters most right now is results.
Why Open-Source R&D?
It was the existing pharmaceutical infrastructure of intellectual property hoarding that left our species so poorly prepared for a pandemic like COVID-19. If we’re going to learn to survive in a future that may contain similar or worse pandemics, we’re going to need to redesign our system of rewarding innovation. Rather than accusing Chinese firms of intellectual property theft, the Western pharmaceutical industry should be building research collaborations. Likewise, the outdated scientific publishing industry slows the speed of innovation by charging researchers to publish their work behind paywalls. We believe that a solution to this crisis can only by achieved by the free and open sharing of scientific research.
Why Diagnosis? Why not work on a cure?
BioFoundry is only PC1. We are unable to handle pathogenic samples and could not countenance putting our wider community at risk. Instead we will obtain a positive control through proper channels and focus on testing. That said, the initial testing of human samples using our kit will likely require self-experimentation.
Who else are we working with?
Sources and groups referenced while writing page: